Antibodies that bind par-2

ABSTRACT

The present invention provides compositions and methods relating to or derived from anti-PAR-2 antibodies. In particular embodiments, the invention provides antibodies that bind human PAR-2, PAR-2-binding fragments and derivatives of such antibodies, and PAR-2-binding polypeptides comprising such fragments. Other embodiments provide nucleic acids encoding such antibodies, antibody fragments and derivatives and polypeptides, cells comprising such polynucleotides, methods of making such antibodies, antibody fragments and derivatives and polypeptides, and methods of using such antibodies, antibody fragments and derivatives and polypeptides, including methods of treating or diagnosing subjects having PAR-2-related disorders or conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/008,736, filed Jan. 18, 2011, now allowed, which is a divisional ofU.S. patent application Ser. No. 11/704,529, filed Feb. 8, 2007, nowU.S. Pat. No. 7,888,482, issued Feb. 15, 2011, which claims the benefitunder 35 U.S.C. 119(e) of U.S. provisional application Ser. No.60/772,456, filed Feb. 10, 2006; all of which are incorporated byreference herein.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledA-1099-US-CNT_seq_listing.txt, created Sep. 27, 2012, which is 23.2 KBin size. The information in the electronic format of the SequenceListing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This application provides compositions and methods relating toanti-PAR-2 antibodies.

BACKGROUND OF THE INVENTION

The Proteinase-activated receptor (PAR) family is a part of theseven-transmembrane G-coupled receptor superfamily. There are currentlyfour known PARs, of which three (PARs-1, -3 and -4) are activated bythrombin; a fourth (PAR-2) is activated by trypsin or mast celltryptase, but not by thrombin. PARs are widely distributed to a varietyof tissues and participate in a number of physiological orpathophysiological phenomena such as platelet aggregation, inflammationand cardiovascular, digestive or respiratory functions.

PARs differ from other receptors in that activation is initiated byproteolytic cleavage of the N terminus of the PAR, which then forms atethered ligand that interacts with the extracellular region (loop 2) ofthe same receptor polypeptide. Cleavage of PAR-2 occurs between the Rand S residues of the protease cleavage domain, SKGRSLIG (amino acids 33through 40 of SEQ ID NO:2), which is conserved between human, murine andrat PAR-2. Peptides that mimic the tethered ligand have been shown tohave agonistic effects on PAR-2 (Saifeddine et al., Br J Pharmacol118(3):521-30 [1996]; McGuire et al., J Pharmacol Exp Ther309(3):1124-31 [2004]).

PAR-2 activates the G-protein-coupled receptor-mediated common signaltransduction pathways, inositol 1,4,5-trisphosphate production andmobilization of Ca(2+), as well as multiple kinase pathways, includingERK, p38MAPK, JNK, and IKK. It is present on epithelial and endothelialcells, myocytes, fibroblasts, immune cells, neurons and glial cells inthe kidney, pancreas, stomach, intestine, airway, skin, bladder andbrain. The protease that activates PAR-2 present during inflammation,and PAR-2 is upregulated by inflammatory factors such as tumour necrosisfactor alpha, interleukin 1 alpha and lipopolysaccharide. Moreover,studies utilizing PAR-2-deficient or-overexpressing mice confirm a rolefor this receptor in inflammation (Schmidlin et al., J. Immunol. 169,5315-5321 [2002]; Ferrell et al., J. Clin. Invest. 111, 35-41 [2003]).Accordingly, there is a need in the art to develop antagonists of PAR-2activation, which will be useful in treating or amelioratinginflammatory conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates that ability of a PAR-2 antibody to antagonize PAR-2activation in a FLIPR assay, using various PAR-2-expressing cells.

FIG. 2 provides a Western blot comparing the binding of various PAR-2antibodies to full-length versus clipped PAR-2/Fc.

FIG. 3 presents Western blot results for an antagonistic PAR-2 antibody(clone 33) as well as an antibody that does not antagonize PAR-2 (clone10).

FIG. 4 compares that ability of several PAR-2 antibodies to antagonizePAR-2 activation in a FLIPR assay.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides an isolated antigenbinding protein that binds to proteinase activated receptor-2 (PAR-2).In another embodiment, the isolated antigen binding protein, when boundto a human PAR-2, inhibits proteolytic cleavage and/or subsequentsignaling through said human PAR-2. In another embodiment, the isolatedantigen binding protein inhibits proteolytic activation of PAR-2 bygreater than about 80%. In another embodiment, the isolated antigenbinding protein binds to full-length PAR-2 and binds to a lesser extentto cleaved PAR-2.

In another aspect of the invention, the isolated antigen binding proteinspecifically binds to the PAR-2 of a non-human primate, a cynomologousmonkey, a chimpanzee, a non-primate mammal, a rodent, a mouse, a rat, ahamster, a guinea pig, a cat, or a dog. In another embodiment, theisolated antigen binding protein comprises: a. a human antibody; b. ahumanized antibody; c. a chimeric antibody; d. a monoclonal antibody; e.a polyclonal antibody; f. a recombinant antibody; g. an antigen-bindingantibody fragment; h. a single chain antibody; i. a diabody; j. atriabody; k. a tetrabody; l. a Fab fragment; m. a F(ab′)₂ fragment; n. adomain antibody; o. an IgD antibody; p. an IgE antibody; q. an IgMantibody; r. an IgG1 antibody; s. an IgG2 antibody; t. an IgG3 antibody;u. an IgG4 antibody; or v. an IgG4 antibody having at least one mutationin a hinge region that alleviates a tendency to form intra-H chaindisulfide bond.

In another aspect, the present invention provides an isolated cell thatsecretes an antigen binding protein that binds PAR-2. In anotherembodiment, the cell is a hybridoma. In another embodiment, the presentinvention provides a method of making an antigen binding protein thatbinds human PAR-2, comprising incubating said isolated cell underconditions that allow it to express said antigen binding protein.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising the antigen binding protein. In one embodiment,the present invention provides a method of treating a condition in asubject comprising administering to said subject said pharmaceuticalcomposition, wherein said condition is treatable by reducing theactivity of PAR-2 in said subject. In another embodiment, said subjectis a human being. In another embodiment, said condition is aninflammatory condition of the skin, joints, gastrointestinal systemand/or airway. In another embodiment, the method further comprisesadministering to said subject a second treatment. In another embodiment,said second treatment is administered to said subject before and/orsimultaneously with and/or after said pharmaceutical composition isadministered to said subject. In another embodiment, said secondtreatment comprises an anti-inflammatory agent. In another embodiment,said second pharmaceutical composition comprises an agent selected fromthe group consisting of non-steroidal anti-inflammatory drugs, steroids,and immunomodulating agents. In another embodiment, said methodcomprises administering to said subject a third treatment.

In another aspect, the present invention provides a method of increasingthe longevity of a subject comprising administering to said subject saidpharmaceutical composition.

In another aspect, the present invention provides a method of decreasingPAR-2 activity in a subject in need thereof comprising administering tosaid subject said pharmaceutical composition.

In another aspect, the present invention provides a method of decreasingPAR-2 signaling in a subject in need thereof comprising administering tosaid subject said pharmaceutical composition.

In another aspect, the present invention provides a method of inhibitingthe proteolytic activation of PAR-2 in a subject in need thereofcomprising administering to said subject said pharmaceuticalcomposition.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions, kits, and methods relatingto molecules that bind to the Proteinase Activated Receptor 2 (“PAR-2”),including molecules that agonize or antagonize PAR-2, such as anti-PAR-2antibodies, antibody fragments, and antibody derivatives, e.g.,antagonistic anti-PAR-2 antibodies, antibody fragments, or antibodyderivatives. Also provided are nucleic acids, and derivatives andfragments thereof, comprising a sequence of nucleotides that encodes allor a portion of a polypeptide that binds to PAR-2, e.g., a nucleic acidencoding all or part of an anti-PAR-2 antibody, antibody fragment, orantibody derivative, plasmids and vectors comprising such nucleic acids,and cells or cell lines comprising such nucleic acids and/or vectors andplasmids. The provided methods include, for example, methods of making,identifying, or isolating molecules that bind to PAR-2, such asanti-PAR-2 antibodies, methods of determining whether a molecule bindsto PAR-2, methods of determining whether a molecule agonizes orantagonizes PAR-2, methods of making compositions, such aspharmaceutical compositions, comprising a molecule that binds to PAR-2,and methods for administering a molecule that binds PAR-2 to a subject,for example, methods for treating a condition mediated by PAR-2, and foragonizing or antagonizing a biological activity of PAR-2, in vivo or invitro.

Polynucleotide and polypeptide sequences are indicated using standardone- or three-letter abbreviations. Unless otherwise indicated, eachpolypeptide sequence has amino termini at the left and a carboxy terminiat the right; each single-stranded nucleic acid sequence, and the topstrand of each double-stranded nucleic acid sequence, has a 5′ terminiat the left and a 3′ termini at the right. A particular polypeptide orpolynucleotide sequence also can be described by explaining how itdiffers from a reference sequence.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, geneticsand protein and nucleic acid chemistry and hybridization describedherein are those well known and commonly used in the art. The methodsand techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al. Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates (1992), and Harlow and Lane Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1990), which are incorporated herein by reference.Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications, as commonly accomplished in the art oras described herein. The terminology used in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those well known and commonly used in the art. Standardtechniques can be used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofpatients.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

The term “isolated molecule” (where the molecule is, for example, apolypeptide, a polynucleotide, or an antibody) is a molecule that byvirtue of its origin or source of derivation (1) is not associated withnaturally associated components that accompany it in its native state,(2) is substantially free of other molecules from the same species (3)is expressed by a cell from a different species, or (4) does not occurin nature. Thus, a molecule that is chemically synthesized, orsynthesized in a cellular system different from the cell from which itnaturally originates, will be “isolated” from its naturally associatedcomponents. A molecule also may be rendered substantially free ofnaturally associated components by isolation, using purificationtechniques well known in the art. Molecule purity or homogeneity may beassayed by a number of means well known in the art. For example, thepurity of a polypeptide sample may be assayed using polyacrylamide gelelectrophoresis and staining of the gel to visualize the polypeptideusing techniques well known in the art. For certain purposes, higherresolution may be provided by using HPLC or other means well known inthe art for purification.

The terms “PAR-2 inhibitor” and “PAR-2 antagonist” are usedinterchangeably. Each is a molecule that detectably inhibits at leastone function of PAR-2. Conversely, a “PAR-2 agonist” is a molecule thatdetectably increases at least one function of PAR-2. The inhibitioncaused by a PAR-2 inhibitor need not be complete so long as it isdetectable using an assay. Any assay of a function of PAR-2 can be used,examples of which are provided herein. Examples of functions of PAR-2that can be inhibited by a PAR-2 inhibitor, or increased by a PAR-2agonist, include protease-activated ligand binding, downstreamsignaling, and so on. Examples of types of PAR-2 inhibitors and PAR-2agonists include, but are not limited to, PAR-2 binding polypeptidessuch as antigen binding proteins (e.g., PAR-2 inhibiting antigen bindingproteins), antibodies, antibody fragments, and antibody derivatives.

The terms “peptide,” “polypeptide” and “protein” each refers to amolecule comprising two or more amino acid residues joined to each otherby peptide bonds. These terms encompass, e.g., native and artificialproteins, protein fragments and polypeptide analogs (such as muteins,variants, and fusion proteins) of a protein sequence as well aspost-translationally, or otherwise covalently or non-covalently,modified proteins. A peptide, polypeptide, or protein may be monomericor polymeric.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion as comparedto a corresponding full-length protein. Fragments can be, for example,at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 50, 70, 80, 90, 100,150 or 200 amino acids in length. Fragments can also be, for example, atmost 1,000, 750, 500, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50,40, 30, 20, 15, 14, 13, 12, 11, or 10 amino acids in length. A fragmentcan further comprise, at either or both of its ends, one or moreadditional amino acids, for example, a sequence of amino acids from adifferent naturally-occurring protein (e.g., an Fc or leucine zipperdomain) or an artificial amino acid sequence (e.g., an artificial linkersequence or a tag protein).

Polypeptides of the invention include polypeptides that have beenmodified in any way and for any reason, for example, to: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties. Analogs include muteins of a polypeptide. Forexample, single or multiple amino acid substitutions (e.g., conservativeamino acid substitutions) may be made in the naturally occurringsequence (e.g., in the portion of the polypeptide outside the domain(s)forming intermolecular contacts. A “conservative amino acidsubstitution” is one that does not substantially change the structuralcharacteristics of the parent sequence (e.g., a replacement amino acidshould not tend to break a helix that occurs in the parent sequence, ordisrupt other types of secondary structure that characterize the parentsequence or are necessary for its functionality). Examples ofart-recognized polypeptide secondary and tertiary structures aredescribed in Proteins, Structures and Molecular Principles (Creighton,Ed., W. H. Freeman and Company, New York (1984)); Introduction toProtein Structure (C. Branden and J. Tooze, eds., Garland Publishing,New York, N.Y. (1991)); and Thornton et at. Nature 354:105 (1991), whichare each incorporated herein by reference.

The present invention also provides non-peptide analogs of PAR-2 bindingpolypeptides. Non-peptide analogs are commonly used in thepharmaceutical industry as drugs with properties analogous to those ofthe template peptide. These types of non-peptide compound are termed“peptide mimetics” or “peptidomimetics,” see, for example, Fauchere, J.Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p. 392 (1985);and Evans et al. J. Med. Chem. 30:1229 (1987), which are incorporatedherein by reference. Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce an equivalenttherapeutic or prophylactic effect. Generally, peptidomimetics arestructurally similar to a paradigm polypeptide (i.e., a polypeptide thathas a desired biochemical property or pharmacological activity), such asa human antibody, but have one or more peptide linkages optionallyreplaced by a linkage selected from the group consisting of: —CH₂NH—,—CH₂S—, —CH₂—CH₂—, —CH═CH— (cis and trans), —COCH₂—, —CH(OH)CH₂—, and—CH₂SO—, by methods well known in the art. Systematic substitution ofone or more amino acids of a consensus sequence with a D-amino acid ofthe same type (e.g., D-lysine in place of L-lysine) may also be used togenerate more stable peptides. In addition, constrained peptidescomprising a consensus sequence or a substantially identical consensussequence variation may be generated by methods known in the art (Rizoand Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference), for example, by adding internal cysteine residues capable offorming intramolecular disulfide bridges which cyclize the peptide.

A “variant” of a polypeptide (e.g., an antibody) comprises an amino acidsequence wherein one or more amino acid residues are inserted into,deleted from and/or substituted into the amino acid sequence relative toanother polypeptide sequence. Variants of the invention include fusionproteins.

A “derivative” of a polypeptide is a polypeptide (e.g., an antibody)that has been chemically modified, e.g., via conjugation to anotherchemical moiety (such as, for example, polyethylene glycol or albumin,e.g., human serum albumin), phosphorylation, and glycosylation. Unlessotherwise indicated, the term “antibody” includes, in addition toantibodies comprising two full-length heavy chains and two full-lengthlight chains, derivatives, variants, fragments, and muteins thereof,examples of which are described below.

An “antigen binding protein” is a protein comprising a portion thatbinds to an antigen and, optionally, a scaffold or framework portionthat allows the antigen binding portion to adopt a conformation thatpromotes binding of the antigen binding protein to the antigen. Examplesof antigen binding proteins include antibodies, antibody fragments(e.g., an antigen binding portion of an antibody), antibody derivatives,and antibody analogs. The antigen binding protein can comprise, forexample, an alternative protein scaffold or artificial scaffold withgrafted CDRs or CDR derivatives. Such scaffolds include, but are notlimited to, antibody-derived scaffolds comprising mutations introducedto, for example, stabilize the three-dimensional structure of theantigen binding protein as well as wholly synthetic scaffoldscomprising, for example, a biocompatible polymer. See, for example,Korndorfer et al., 2003, Proteins: Structure, Function, andBioinformatics, Volume 53, Issue 1:121-129; Roque et al., 2004,Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics(“PAMs”) can be used, as well as scaffolds based on antibody mimeticsutilizing fibronection components as a scaffold.

An antigen binding protein can have, for example, the structure of anaturally occurring immunoglobulin. An “immunoglobulin” is a tetramericmolecule. In a naturally occurring immunoglobulin, each tetramer iscomposed of two identical pairs of polypeptide chains, each pair havingone “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa and lambda light chains. Heavy chains areclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. See generally,Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)) (incorporated by reference in its entirety for all purposes).The variable regions of each light/heavy chain pair form the antibodybinding site such that an intact immunoglobulin has two binding sites.

Naturally occurring immunoglobulin chains exhibit the same generalstructure of relatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor CDRs. From N-terminus to C-terminus, both light and heavy chainscomprise the domains FRE CDR1, FR2, CDR2, FR3, CDR3 and FR4. Theassignment of amino acids to each domain is in accordance with thedefinitions of Kabat et al. in Sequences of Proteins of ImmunologicalInterest, 5^(th) Ed., US Dept. of Health and Human Services, PHS, NIH,NIH Publication no. 91-3242, 1991.

Naturally occurring antibodies can be obtained from sources such asserum or plasma that contain immunoglobulins having varied antigenicspecificity. If such antibodies are subjected to affinity purification,they can be enriched for a particular antigenic specificity. Suchenriched preparations of antibodies usually are made of less than about10% antibody having specific binding activity for the particularantigen. Antibodies prepared in this manner are often referred to as“monospecific.”

An “antibody” refers to an intact immunoglobulin or to an antigenbinding portion thereof that competes with the intact antibody forspecific binding, unless otherwise specified. Antigen binding portionsmay be produced by recombinant DNA techniques or by enzymatic orchemical cleavage of intact antibodies. Antigen binding portionsinclude, inter alia, Fab, Fab′, F(ab′)₂, Fv, domain antibodies (dAbs),and complementarity determining region (CDR) fragments, single-chainantibodies (scFv), chimeric antibodies, diabodies, triabodies,tetrabodies, and polypeptides that contain at least a portion of animmunoglobulin that is sufficient to confer specific antigen binding tothe polypeptide.

A Fab fragment is a monovalent fragment having the V_(L), V_(H), C_(L)and C_(H)1 domains; a F(ab′)₂ fragment is a bivalent fragment having twoFab fragments linked by a disulfide bridge at the hinge region; a Fdfragment has the V_(H) and C_(H)1 domains; an Fv fragment has the V_(L)and V_(H) domains of a single arm of an antibody; and a dAb fragment hasa V_(H) domain, a V_(L) domain, or an antigen-binding fragment of aV_(H) or V_(L) domain (U.S. Pat. Nos. 6,846,634, 6,696,245, US App. Pub.No. 05/0202512, 04/0202995, 04/0038291, 04/0009507, 03/0039958, Ward etal., Nature 341:544-546, 1989).

A single-chain antibody (scFv) is an antibody in which a V_(L) and aV_(H) region are joined via a linker (e.g., a synthetic sequence ofamino acid residues) to form a continuous protein chain wherein thelinker is long enough to allow the protein chain to fold back on itselfand form a monovalent antigen binding site (see, e.g., Bird et al.,1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci.USA 85:5879-83). Diabodies are bivalent antibodies comprising twopolypeptide chains, wherein each polypeptide chain comprises V_(H) andV_(L) domains joined by a linker that is too short to allow for pairingbetween two domains on the same chain, thus allowing each domain to pairwith a complementary domain on another polypeptide chain (see, e.g.,Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-48, and Poljaket al., 1994, Structure 2:1121-23). If the two polypeptide chains of adiabody are identical, then a diabody resulting from their pairing willhave two identical antigen binding sites. Polypeptide chains havingdifferent sequences can be used to make a diabody with two differentantigen binding sites. Similarly, tribodies and tetrabodies areantibodies comprising three and four polypeptide chains, respectively,and forming three and four antigen binding sites, respectively, whichcan be the same or different.

Complementarity determining regions (CDRs) and framework regions (FR) ofa given antibody may be identified using the system described by Kabatet al. in Sequences of Proteins of Immunological Interest, 5th Ed., USDept. of Health and Human Services, PHS, NIH, NIH Publication no.91-3242, 1991. One or more CDRs may be incorporated into a moleculeeither covalently or noncovalently to make it an antigen bindingprotein. An antigen binding protein may incorporate the CDR(s) as partof a larger polypeptide chain, may covalently link the CDR(s) to anotherpolypeptide chain, or may incorporate the CDR(s) noncovalently. The CDRspermit the antigen binding protein to specifically bind to a particularantigen of interest.

An antigen binding protein may have one or more binding sites. If thereis more than one binding site, the binding sites may be identical to oneanother or may be different. For example, a naturally occurring humanimmunoglobulin typically has two identical binding sites, while a“bispecific” or “bifunctional” antibody has two different binding sites.

The term “human antibody” includes all antibodies that have one or morevariable and constant regions derived from human immunoglobulinsequences. In one embodiment, all of the variable and constant domainsare derived from human immunoglobulin sequences (a fully humanantibody). These antibodies may be prepared in a variety of ways,examples of which are described below, including through theimmunization with an antigen of interest of a mouse that is geneticallymodified to express antibodies derived from human heavy and/or lightchain-encoding genes.

A humanized antibody has a sequence that differs from the sequence of anantibody derived from a non-human species by one or more amino acidsubstitutions, deletions, and/or additions, such that the humanizedantibody is less likely to induce an immune response, and/or induces aless severe immune response, as compared to the non-human speciesantibody, when it is administered to a human subject. In one embodiment,certain amino acids in the framework and constant domains of the heavyand/or light chains of the non-human species antibody are mutated toproduce the humanized antibody. In another embodiment, the constantdomain(s) from a human antibody are fused to the variable domain(s) of anon-human species. In another embodiment, one or more amino acidresidues in one or more CDR sequences of a non-human antibody arechanged to reduce the likely immunogenicity of the non-human antibodywhen it is administered to a human subject, wherein the changed aminoacid residues either are not critical for immunospecific binding of theantibody to its antigen, or the changes to the amino acid sequence thatare made are conservative changes, such that the binding of thehumanized antibody to the antigen is not significantly worse than thebinding of the non-human antibody to the antigen. Examples of how tomake humanized antibodies may be found in U.S. Pat. Nos. 6,054,297,5,886,152 and 5,877,293.

The term “chimeric antibody” refers to an antibody that contains one ormore regions from one antibody and one or more regions from one or moreother antibodies. In one embodiment, one or more of the CDRs are derivedfrom a human anti-PAR-2 antibody. In another embodiment, all of the CDRsare derived from a human anti-PAR-2 antibody. In another embodiment, theCDRs from more than one human anti-PAR-2 antibodies are mixed andmatched in a chimeric antibody. For instance, a chimeric antibody maycomprise a CDR1 from the light chain of a first human anti-PAR-2antibody, a CDR2 and a CDR3 from the light chain of a second humananti-PAR-2 antibody, and the CDRs from the heavy chain from a thirdanti-PAR-2 antibody. Further, the framework regions may be derived fromone of the same anti-PAR-2 antibodies, from one or more differentantibodies, such as a human antibody, or from a humanized antibody. Inone example of a chimeric antibody, a portion of the heavy and/or lightchain is identical with, homologous to, or derived from an antibody froma particular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is/are identical with,homologous to, or derived from an antibody(-ies) from another species orbelonging to another antibody class or subclass. Also included arefragments of such antibodies that exhibit the desired biologicalactivity (i.e., the ability to specifically bind PAR-2). See, e.g., U.S.Pat. No. 4,816,567 and Morrison, 1985, Science 229:1202-07.

A “neutralizing antibody” or “an inhibitory antibody” is an antibodythat inhibits the proteolytic activation of PAR-2 when an excess of theanti-PAR-2 antibody reduces the amount of activation by at least about20% using an assay such as those described herein in the Examples. Invarious embodiments, the antibody reduces the amount of amount ofproteolytic activation of PAR-2 by at least 30%, 40%, 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 97%, 99%, and 99.9%.

Fragments or analogs of antibodies can be readily prepared by those ofordinary skill in the art following the teachings of this specificationand using techniques well-known in the art. Preferred amino- andcarboxy-termini of fragments or analogs occur near boundaries offunctional domains. Structural and functional domains can be identifiedby comparison of the nucleotide and/or amino acid sequence data topublic or proprietary sequence databases. Computerized comparisonmethods can be used to identify sequence motifs or predicted proteinconformation domains that occur in other proteins of known structureand/or function. Methods to identify protein sequences that fold into aknown three-dimensional structure are known. See, e.g., Bowie et al.,1991, Science 253:164.

A “CDR grafted antibody” is an antibody comprising one or more CDRsderived from an antibody of a particular species or isotype and theframework of another antibody of the same or different species orisotype.

A “multi-specific antibody” is an antibody that recognizes more than oneepitope on one or more antigens. A subclass of this type of antibody isa “bi-specific antibody” which recognizes two distinct epitopes on thesame or different antigens.

An antigen binding protein “specifically binds” to an antigen (e.g.,human PAR-2) if it binds to the antigen with a dissociation constant of1 nanomolar or less.

An “antigen binding domain,” “antigen binding region,” or “antigenbinding site” is a portion of an antigen binding protein that containsamino acid residues (or other moieties) that interact with an antigenand contribute to the antigen binding protein's specificity and affinityfor the antigen. For an antibody that specifically binds to its antigen,this will include at least part of at least one of its CDR domains.

An “epitope” is the portion of a molecule that is bound by an antigenbinding protein (e.g., by an antibody). An epitope can comprisenon-contiguous portions of the molecule (e.g., in a polypeptide, aminoacid residues that are not contiguous in the polypeptide's primarysequence but that, in the context of the polypeptide's tertiary andquaternary structure, are near enough to each other to be bound by anantigen binding protein).

The “percent identity” of two polynucleotide or two polypeptidesequences is determined by comparing the sequences using the GAPcomputer program (a part of the GCG Wisconsin Package, version 10.3(Accelrys, San Diego, Calif.)) using its default parameters.

The terms “polynucleotide,” “oligonucleotide” and “nucleic acid” areused interchangeably throughout and include DNA molecules (e.g., cDNA orgenomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNAgenerated using nucleotide analogs (e.g., peptide nucleic acids andnon-naturally occurring nucleotide analogs), and hybrids thereof. Thenucleic acid molecule can be single-stranded or double-stranded. In oneembodiment, the nucleic acid molecules of the invention comprise acontiguous open reading frame encoding an antibody, or a fragment,derivative, mutein, or variant thereof, of the invention.

Two single-stranded polynucleotides are “the complement” of each otherif their sequences can be aligned in an anti-parallel orientation suchthat every nucleotide in one polynucleotide is opposite itscomplementary nucleotide in the other polynucleotide, without theintroduction of gaps, and without unpaired nucleotides at the 5′ or the3′ end of either sequence. A polynucleotide is “complementary” toanother polynucleotide if the two polynucleotides can hybridize to oneanother under moderately stringent conditions. Thus, a polynucleotidecan be complementary to another polynucleotide without being itscomplement.

A “vector” is a nucleic acid that can be used to introduce anothernucleic acid linked to it into a cell. One type of vector is a“plasmid,” which refers to a linear or circular double stranded DNAmolecule into which additional nucleic acid segments can be ligated.Another type of vector is a viral vector (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), whereinadditional DNA segments can be introduced into the viral genome. Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors comprising a bacterialorigin of replication and episomal mammalian vectors). Other vectors(e.g., non-episomal mammalian vectors) are integrated into the genome ofa host cell upon introduction into the host cell, and thereby arereplicated along with the host genome. An “expression vector” is a typeof vector that can direct the expression of a chosen polynucleotide.

A nucleotide sequence is “operably linked” to a regulatory sequence ifthe regulatory sequence affects the expression (e.g., the level, timing,or location of expression) of the nucleotide sequence. A “regulatorysequence” is a nucleic acid that affects the expression (e.g., thelevel, timing, or location of expression) of a nucleic acid to which itis operably linked. The regulatory sequence can, for example, exert itseffects directly on the regulated nucleic acid, or through the action ofone or more other molecules (e.g., polypeptides that bind to theregulatory sequence and/or the nucleic acid). Examples of regulatorysequences include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Further examples of regulatorysequences are described in, for example, Goeddel, 1990, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.and Baron et al., 1995, Nucleic Acids Res. 23:3605-06.

A “host cell” is a cell that can be used to express a nucleic acid,e.g., a nucleic acid of the invention. A host cell can be a prokaryote,for example, E. coli, or it can be a eukaryote, for example, asingle-celled eukaryote (e.g., a yeast or other fungus), a plant cell(e.g., a tobacco or tomato plant cell), an animal cell (e.g., a humancell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or aninsect cell) or a hybridoma. Examples of host cells include the COS-7line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al., 1981,Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinesehamster ovary (CHO) cells or their derivatives such as Veggie CHO andrelated cell lines which grow in serum-free media (see Rasmussen et al.,1998, Cytotechnology 28:31) or CHO strain DX-B11, which is deficient inDHFR (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20),HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1/EBNA cell line derivedfrom the African green monkey kidney cell line CV1 (ATCC CCL 70) (seeMcMahan et al., 1991, EMBO J. 10:2821), human embryonic kidney cellssuch as 293, 293 EBNA or MSR 293, human epidermal A431 cells, humanCo1o205 cells, other transformed primate cell lines, normal diploidcells, cell strains derived from in vitro culture of primary tissue,primary explants, HL-60, U937, HaK or Jurkat cells. Typically, a hostcell is a cultured cell that can be transformed or transfected with apolypeptide-encoding nucleic acid, which can then be expressed in thehost cell. The phrase “recombinant host cell” can be used to denote ahost cell that has been transformed or transfected with a nucleic acidto be expressed. A host cell also can be a cell that comprises thenucleic acid but does not express it at a desired level unless aregulatory sequence is introduced into the host cell such that itbecomes operably linked with the nucleic acid. It is understood that theterm host cell refers not only to the particular subject cell but alsoto the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to, e.g., mutationor environmental influence, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

PAR-2 Antigen Binding Proteins

In one aspect, the present invention provides antigen binding proteins(e.g., antibodies, antibody fragments, antibody derivatives, antibodymuteins, and antibody variants) that bind to PAR-2, e.g., human PAR-2.

Antigen binding proteins in accordance with the present inventioninclude antigen binding proteins that inhibit a biological activity ofPAR-2. Examples of such biological activities include activation ofG-protein-coupled receptor-mediated common signal transduction pathwayssuch as inositol 1,4,5-trisphosphate production and mobilization ofCa(2+), and activation of multiple kinase pathways, including ERK,p38MAPK, JNK, and IKK. Other biological activities include thosemediated by PAR-2 in vivo, such as the response to trauma andinflammation; in particular, PAR2 is involved in the cardiovascular,pulmonary and gastrointestinal systems, where it controls inflammationand nociception (perception of pain). PAR-2 activation also plays a rolein the inflammatory response, chronic activation of which can lead todisease conditions.

Different antigen binding proteins may bind to different domains orepitopes of PAR-2 or act by different mechanisms of action. Examplesinclude but are not limited to antigen binding proteins that interferewith proteolytic activation of PAR-2 or that inhibit signaltransduction. The site of action may be, for example, intracellular(e.g., by interfering with an intracellular signaling cascade) orextracellular. An antigen binding protein need not completely inhibitPAR-2 induced activity to find use in the present invention; rather,antigen binding proteins that reduce a particular activity of PAR-2 arecontemplated for use as well. (Discussions herein of particularmechanisms of action for PAR-2-binding antigen binding proteins intreating particular diseases are illustrative only, and the methodspresented herein are not bound thereby.)

Other derivatives of anti-PAR-2 antibodies within the scope of thisinvention include covalent or aggregative conjugates of anti-PAR-2antibodies, or fragments thereof, with other proteins or polypeptides,such as by expression of recombinant fusion proteins comprisingheterologous polypeptides fused to the N-terminus or C-terminus of ananti-PAR-2 antibody polypeptide. For example, the conjugated peptide maybe a heterologous signal (or leader) polypeptide, e.g., the yeastalpha-factor leader, or a peptide such as an epitope tag. Antigenbinding protein-containing fusion proteins can comprise peptides addedto facilitate purification or identification of antigen binding protein(e.g., poly-His). An antigen binding protein also can be linked to theFLAG peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK) (SEQ ID NO:7) asdescribed in Hopp et al., Bio/Technology 6:1204, 1988, and U.S. Patent5,011,912. The FLAG peptide is highly antigenic and provides an epitopereversibly bound by a specific monoclonal antibody (mAb), enabling rapidassay and facile purification of expressed recombinant protein. Reagentsuseful for preparing fusion proteins in which the FLAG peptide is fusedto a given polypeptide are commercially available (Sigma, St. Louis,Mo.).

Oligomers that contain one or more antigen binding proteins may beemployed as PAR-2 antagonists. Oligomers may be in the form ofcovalently-linked or non-covalently-linked dimers, trimers, or higheroligomers. Oligomers comprising two or more antigen binding protein arecontemplated for use, with one example being a homodimer. Otheroligomers include heterodimers, homotrimers, heterotrimers,homotetramers, heterotetramers, etc.

One embodiment is directed to oligomers comprising multiple antigenbinding proteins joined via covalent or non-covalent interactionsbetween peptide moieties fused to the antigen binding proteins. Suchpeptides may be peptide linkers (spacers), or peptides that have theproperty of promoting oligomerization. Leucine zippers and certainpolypeptides derived from antibodies are among the peptides that canpromote oligomerization of antigen binding proteins attached thereto, asdescribed in more detail below.

In particular embodiments, the oligomers comprise from two to fourantigen binding proteins. The antigen binding proteins of the oligomermay be in any form, such as any of the forms described above, e.g.,variants or fragments. Preferably, the oligomers comprise antigenbinding proteins that have PAR-2 binding activity.

In one embodiment, an oligomer is prepared using polypeptides derivedfrom immunoglobulins. Preparation of fusion proteins comprising certainheterologous polypeptides fused to various portions of antibody-derivedpolypeptides (including the Fc domain) has been described, e.g., byAshkenazi et al., 1991, PNAS USA 88:10535; Byrn et al., 1990, Nature344:677; and Hollenbaugh et al., 1992 “Construction of ImmunoglobulinFusion Proteins”, in Current Protocols in Immunology, Suppl. 4, pages10.19.1 -10.19.11.

One embodiment of the present invention is directed to a dimercomprising two fusion proteins created by fusing a PAR-2 bindingfragment of an anti-PAR-2 antibody to the Fc region of an antibody. Thedimer can be made by, for example, inserting a gene fusion encoding thefusion protein into an appropriate expression vector, expressing thegene fusion in host cells transformed with the recombinant expressionvector, and allowing the expressed fusion protein to assemble much likeantibody molecules, whereupon interchain disulfide bonds form betweenthe Fc moieties to yield the dimer.

The term “Fc polypeptide” as used herein includes native and muteinforms of polypeptides derived from the Fc region of an antibody.Truncated forms of such polypeptides containing the hinge region thatpromotes dimerization also are included. Fusion proteins comprising Fcmoieties (and oligomers formed therefrom) offer the advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns.

One suitable Fc polypeptide, described in PCT application WO 93/10151(hereby incorporated by reference), is a single chain polypeptideextending from the N-terminal hinge region to the native C-terminus ofthe Fc region of a human IgG1 antibody. Another useful Fc polypeptide isthe Fc mutein described in U.S. Pat. No. 5,457,035 and in Baum et al.,1994, EMBO J. 13:3992-4001. The amino acid sequence of this mutein isidentical to that of the native Fc sequence presented in WO 93/10151,except that amino acid 19 has been changed from Leu to Ala, amino acid20 has been changed from Leu to Glu, and amino acid 22 has been changedfrom Gly to Ala. The mutein exhibits reduced affinity for Fc receptors.

In other embodiments, the variable portion of the heavy and/or lightchains of an anti-PAR-2 antibody may be substituted for the variableportion of an antibody heavy and/or light chain.

Alternatively, the oligomer is a fusion protein comprising multipleantigen binding proteins, with or without peptide linkers (spacerpeptides). Among the suitable peptide linkers are those described inU.S. Pat. Nos. 4,751,180 and 4,935,233.

Another method for preparing oligomeric antigen binding proteinsinvolves use of a leucine zipper.

Leucine zipper domains are peptides that promote oligomerization of theproteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins (Landschulz et al., 1988,Science 240:1759), and have since been found in a variety of differentproteins. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize. Examples ofleucine zipper domains suitable for producing soluble oligomericproteins are described in PCT application WO 94/10308, and the leucinezipper derived from lung surfactant protein D (SPD) described in Hoppeet al., 1994, FEBS Letters 344:191, hereby incorporated by reference.The use of a modified leucine zipper that allows for stabletrimerization of a heterologous protein fused thereto is described inFanslow et al., 1994, Semin Immunol 6:267-78. In one approach,recombinant fusion proteins comprising an anti-PAR-2 antibody fragmentor derivative fused to a leucine zipper peptide are expressed insuitable host cells, and the soluble oligomeric anti-PAR-2 antibodyfragments or derivatives that form are recovered from the culturesupernatant.

In one aspect, the present invention provides antigen binding proteinsthat interfere with the proteolytic activation of a PAR-2. Such antigenbinding proteins can be made against PAR-2, or a fragment, variant orderivative thereof, and screened in conventional assays for the abilityto interfere with proteolytic activation of PAR-2. Examples of suitableassays are assays that test the antigen binding proteins for the abilityto inhibit proteolytic activation of cells expressing PAR-2, or thattest antigen binding proteins for the ability to reduce a biological orcellular response that results from the proteolytic activation of cellsurface PAR-2 receptors. Additional assays that test the antigen bindingproteins include those that qualitatively or quantitatively compare thebinding of an antigen binding protein to a full-length, mature PAR-2polypeptide to the binding of a proteolytically cleaved PAR-2polypeptide, several examples of which are disclosed herein.

In another aspect, the present invention provides an antigen bindingprotein that demonstrates species selectivity. In one embodiment, theantigen binding protein binds to one or more mammalian PAR-2, forexample, to human PAR-2 and one or more of mouse, rat, guinea pig,hamster, gerbil, cat, rabbit, dog, goat, sheep, cow, horse, camel, andnon-human primate PAR-2. In another embodiment, the antigen bindingprotein binds to one or more primate PAR-2, for example, to human PAR-2and one or more of cynomologous, marmoset, rhesus, and chimpanzee PAR-2.In another embodiment, the antigen binding protein binds specifically tohuman, cynomologous, marmoset, rhesus, or chimpanzee PAR-2. In anotherembodiment, the antigen binding protein does not bind to one or more ofmouse, rat, guinea pig, hamster, gerbil, cat, rabbit, dog, goat, sheep,cow, horse, camel, and non-human primate PAR-2. In another embodiment,the antigen binding protein does not bind to a New World monkey speciessuch as a marmoset. In another embodiment, the antigen binding proteindoes not exhibit specific binding to any naturally occurring proteinother than PAR-2. In another embodiment, the antigen binding proteindoes not exhibit specific binding to any naturally occurring proteinother than mammalian PAR-2. In another embodiment, the antigen bindingprotein does not exhibit specific binding to any naturally occurringprotein other than primate PAR-2. In another embodiment, the antigenbinding protein does not exhibit specific binding to any naturallyoccurring protein other than human PAR-2. In another embodiment, theantigen binding protein specifically binds to mouse, rat, cynomolgusmonkey, and human PAR-2. In another embodiment, the antigen bindingprotein specifically binds to mouse, rat, cynomolgus monkey, and humanPAR-2 with a similar binding affinity In another embodiment, the antigenbinding protein blocks binding of proteolytic activation of mouse, rat,cynomolgus monkey, and human PAR-2. In another embodiment, the antigenbinding protein has a similar IC50 against mouse, rat, cynomolgusmonkey, and human PAR-2 in a Ca2+ mobilization assay.

One may determine the selectivity of an antigen binding protein for aPAR-2 using methods well known in the art and following the teachings ofthe specification. For example, one may determine the selectivity usingWestern blot, FACS, ELISA or RIA.

In another aspect, the present invention provides a PAR-2 bindingantigen binding protein (for example, an anti-PAR-2 antibody), that hasone or more of the following characteristics: binds to both human andmurine PAR-2, inhibits the proteolytic activation of human PAR-2,inhibits the proteolytic activation of murine PAR-2, binds to or nearthe proteolytic cleavage site of PAR-2, causes relatively littledown-regulation of cell-surface expressed PAR-2.

Antigen-binding fragments of antigen binding proteins of the inventionmay be produced by conventional techniques. Examples of such fragmentsinclude, but are not limited to, Fab and F(ab′)₂ fragments. Antibodyfragments and derivatives produced by genetic engineering techniquesalso are contemplated.

Additional embodiments include chimeric antibodies, e.g., humanizedversions of non-human (e.g., murine) monoclonal antibodies. Suchhumanized antibodies may be prepared by known techniques, and offer theadvantage of reduced immunogenicity when the antibodies are administeredto humans. In one embodiment, a humanized monoclonal antibody comprisesthe variable domain of a murine antibody (or all or part of the antigenbinding site thereof) and a constant domain derived from a humanantibody. Alternatively, a humanized antibody fragment may comprise theantigen binding site of a murine monoclonal antibody and a variabledomain fragment (lacking the antigen-binding site) derived from a humanantibody. Procedures for the production of chimeric and furtherengineered monoclonal antibodies include those described in Riechmann etal., 1988, Nature 332:323, Liu et al., 1987, Proc. Nat. Acad. Sci. USA84:3439, Larrick et al., 1989, Bio/Technology 7:934, and Winter et al.,1993, TIPS 14:139. In one embodiment, the chimeric antibody is a CDRgrafted antibody. Techniques for humanizing antibodies are discussed in,e.g., U.S. patent application Ser. No. 10/194,975 (published Feb. 27,2003), U.S. Pat. Nos. 5,869,619, 5,225,539, 5,821,337, 5,859,205, Padlanet al., 1995, FASEB J. 9:133-39, and Tamura et al., 2000, J. Immunol164:1432-41.

Procedures have been developed for generating human or partially humanantibodies in non-human animals. For example, mice in which one or moreendogenous immunoglobulin genes have been inactivated by various meanshave been prepared. Human immunoglobulin genes have been introduced intothe mice to replace the inactivated mouse genes. Antibodies produced inthe animal incorporate human immunoglobulin polypeptide chains encodedby the human genetic material introduced into the animal. In oneembodiment, a non-human animal, such as a transgenic mouse, is immunizedwith a PAR-2 polypeptide, such that antibodies directed against thePAR-2 polypeptide are generated in the animal. One example of a suitableimmunogen is a soluble human PAR-2, such as a polypeptide comprising theproteolytic cleavage site of PAR-2, or other immunogenic fragment PAR-2.Another example of a suitable immunogen is cells expressing high levelsof PAR-2, or cell membrane preparations therefrom. Examples oftechniques for production and use of transgenic animals for theproduction of human or partially human antibodies are described in U.S.Pat. Nos. 5,814,318, 5,569,825, and 5,545,806, Davis et al., 2003,Production of human antibodies from transgenic mice in Lo, ed. AntibodyEngineering: Methods and Protocols, Humana Press, NJ:191-200, Kellermannet al., 2002, Curr Opin Biotechnol. 13:593-97, Russel et al., 2000,Infect Immun. 68:1820-26, Gallo et al., 2000, Eur J Immun. 30:534-40,Davis et al., 1999, Cancer Metastasis Rev. 18:421-25, Green, 1999, JImmunol Methods. 231:11-23, Jakobovits, 1998, Advanced Drug DeliveryReviews 31:33-42, Green et al., 1998, J Exp Med. 188:483-95, JakobovitsA, 1998, Exp. Opin. Invest. Drugs. 7:607-14, Tsuda et al., 1997,Genomics. 42:413-21, Mendez et al., 1997, Nat Genet. 15:146-56,Jakobovits, 1994, Curr Biol. 4:761-63, Arbones et al., 1994, Immunity1:247-60, Green et al., 1994, Nat Genet. 7:13-21, Jakobovits et al.,1993, Nature. 362:255-58, Jakobovits et al., 1993, Proc Natl Acad SciUSA. 90:2551-55. Chen, J., M. Trounstine, F. W. Alt, F. Young, C.Kurahara, J. Loring, D. Huszar. “Immunoglobulin gene rearrangement in Bcell deficient mice generated by targeted deletion of the J H locus.”International Immunology 5 (1993): 647-656, Choi et al., 1993, NatureGenetics 4: 117-23, Fishwild et al., 1996, Nature Biotechnology 14:845-51, Harding et al., 1995, Annals of the New York Academy ofSciences, Lonberg et al., 1994, Nature 368: 856-59, Lonberg, 1994,Transgenic Approaches to Human Monoclonal Antibodies in Handbook ofExperimental Pharmacology 113: 49-101, Lonberg et al., 1995, InternalReview of Immunology 13: 65-93, Neuberger, 1996, Nature Biotechnology14: 826, Taylor et al., 1992, Nucleic Acids Research 20: 6287-95, Tayloret al., 1994, International Immunology 6: 579-91, Tomizuka et al., 1997,Nature Genetics 16: 133-43, Tomizuka et al., 2000, Proceedings of theNational Academy of Sciences USA 97: 722-27, Tuaillon et al., 1993,Proceedings of the National Academy of Sciences USA 90: 3720-24, andTuaillon et al., 1994, Journal of Immunology 152: 2912-20.

In another aspect, the present invention provides monoclonal antibodiesthat bind to PAR-2. Monoclonal antibodies may be produced using anytechnique known in the art, e.g., by immortalizing spleen cellsharvested from the transgenic animal after completion of theimmunization schedule. The spleen cells can be immortalized using anytechnique known in the art, e.g., by fusing them with myeloma cells toproduce hybridomas. Myeloma cells for use in hybridoma-producing fusionprocedures preferably are non-antibody-producing, have high fusionefficiency, and enzyme deficiencies that render them incapable ofgrowing in certain selective media which support the growth of only thedesired fused cells (hybridomas). Examples of suitable cell lines foruse in mouse fusions include Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul;examples of cell lines used in rat fusions include R210.RCY3, Y3-Ag1.2.3, IR983F and 4B210. Other cell lines useful for cell fusions areU-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6.

In one embodiment, a hybridoma cell line is produced by immunizing ananimal (e.g., a transgenic animal having human immunoglobulin sequences)with a PAR-2 immunogen; harvesting spleen cells from the immunizedanimal; fusing the harvested spleen cells to a myeloma cell line,thereby generating hybridoma cells; establishing hybridoma cell linesfrom the hybridoma cells, and identifying a hybridoma cell line thatproduces an antibody that binds a PAR-2 polypeptide. Such hybridoma celllines, and anti-PAR-2 monoclonal antibodies produced by them, areencompassed by the present invention.

Monoclonal antibodies secreted by a hybridoma cell line can be purifiedusing any technique known in the art. Hybridomas or mAbs may be furtherscreened to identify mAbs with particular properties, such as theability to block a PAR-2 induced activity. Examples of such screens areprovided in the examples below.

Monoclonal antibodies can also be produced using a process referred toas genetic immunization. For example, a nucleic acid encoding theantigen of interest can be incorporated into a viral vector (such as anadenoviral vector). The resulting vector is then used to develop animmune response against the antigen of interest in a suitable hostanimal (for example, a non-obese diabetic, or NOD, mouse). Thistechniques is substantially described by Ritter et al., Biodrugs 16(1):3-10 (2002), the disclosure of which is incorporated by referenceherein.

Molecular evolution of the complementarity determining regions (CDRs) inthe center of the antibody binding site also has been used to isolateantibodies with increased affinity, for example, antibodies havingincreased affinity for c-erbB-2, as described by Schier et al., 1996, J.Mol. Biol. 263:551. Accordingly, such techniques are useful in preparingantibodies to PAR-2.

Antigen binding proteins directed against a PAR-2 can be used, forexample, in assays to detect the presence of PAR-2 polypeptides, eitherin vitro or in vivo. The antigen binding proteins also may be employedin purifying PAR-2 proteins by immunoaffinity chromatography. Thoseantigen binding proteins that additionally can block proteolyticactivation of PAR-2 may be used to inhibit a biological activity thatresults from such binding. Blocking antigen binding proteins can be usedin the methods of the present invention. Such antigen binding proteinsthat function as PAR-2 antagonists may be employed in treating anyPAR-2-induced condition, including but not limited to inflammatoryconditions. In one embodiment, a human anti-PAR-2 monoclonal antibodygenerated by procedures involving immunization of transgenic mice isemployed in treating such conditions.

Antigen binding proteins may be employed in an in vitro procedure, oradministered in vivo to inhibit a PAR-2-induced biological activity.Disorders caused or exacerbated (directly or indirectly) by theproteolytic activation of PAR-2, examples of which are provided herein,thus may be treated. In one embodiment, the present invention provides atherapeutic method comprising in vivo administration of a PAR-2 blockingantigen binding protein to a mammal in need thereof in an amounteffective for reducing a PAR-2-induced biological activity.

Antigen binding proteins of the invention include partially human andfully human monoclonal antibodies that inhibit a biological activity ofPAR-2. One embodiment is directed to a human monoclonal antibody that atleast partially blocks proteolytic activation of human PAR-2. In oneembodiment, the antibodies are generated by immunizing a transgenicmouse with a PAR-2 immunogen. In another embodiment, the immunogen is ahuman PAR-2 polypeptide (e.g., a soluble fragment comprising all or partof the PAR-2 cleavage site). Hybridoma cell lines derived from suchimmunized mice, wherein the hybridoma secretes a monoclonal antibodythat binds PAR-2, also are provided herein.

Although human, partially human, or humanized antibodies will besuitable for many applications, particularly those involvingadministration of the antibody to a human subject, other types ofantigen binding proteins will be suitable for certain applications. Thenon-human antibodies of the invention can be, for example, derived fromany antibody-producing animal, such as mouse, rat, rabbit, goat, donkey,or non-human primate (such as monkey (e.g., cynomologous or rhesusmonkey) or ape (e.g., chimpanzee)). Non-human antibodies of theinvention can be used, for example, in in vitro and cell-culture basedapplications, or any other application where an immune response to theantibody of the invention does not occur, is insignificant, can beprevented, is not a concern, or is desired. In one embodiment, anon-human antibody of the invention is administered to a non-humansubject. In another embodiment, the non-human antibody does not elicitan immune response in the non-human subject. In another embodiment, thenon-human antibody is from the same species as the non-human subject,e.g., a mouse antibody of the invention is administered to a mouse. Anantibody from a particular species can be made by, for example,immunizing an animal of that species with the desired immunogen (e.g., asoluble PAR-2 polypeptide) or using an artificial system for generatingantibodies of that species (e.g., a bacterial or phage display-basedsystem for generating antibodies of a particular species), or byconverting an antibody from one species into an antibody from anotherspecies by replacing, e.g., the constant region of the antibody with aconstant region from the other species, or by replacing one or moreamino acid residues of the antibody so that it more closely resemblesthe sequence of an antibody from the other species. In one embodiment,the antibody is a chimeric antibody comprising amino acid sequencesderived from antibodies from two or more different species.

Antigen binding proteins may be prepared by any of a number ofconventional techniques. For example, they may be purified from cellsthat naturally express them (e.g., an antibody can be purified from ahybridoma that produces it), or produced in recombinant expressionsystems, using any technique known in the art. See, for example,Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Kennet et al. (eds.), Plenum Press, New York (1980); andAntibodies: A Laboratory Manual, Harlow and Land (eds.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).

Any expression system known in the art can be used to make therecombinant polypeptides of the invention. In general, host cells aretransformed with a recombinant expression vector that comprises DNAencoding a desired polypeptide. Among the host cells that may beemployed are prokaryotes, yeast or higher eukaryotic cells. Prokaryotesinclude gram negative or gram positive organisms, for example E. coli orbacilli. Higher eukaryotic cells include insect cells and establishedcell lines of mammalian origin. Examples of suitable mammalian host celllines include the COS-7 line of monkey kidney cells (ATCC CRL 1651)(Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK(ATCC CRL 10) cell lines, and the CVI/EBNA cell line derived from theAfrican green monkey kidney cell line CVI (ATCC CCL 70) as described byMcMahan et al., 1991, EMBO J. 10: 2821. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described by Pouwels et al. (Cloning Vectors: ALaboratory Manual, Elsevier, N.Y., 1985).

The transformed cells can be cultured under conditions that promoteexpression of the polypeptide, and the polypeptide recovered byconventional protein purification procedures. One such purificationprocedure includes the use of affinity chromatography, e.g., over amatrix having all or a portion (e.g., the extracellular domain) of PAR-2bound thereto. Polypeptides contemplated for use herein includesubstantially homogeneous recombinant mammalian anti-PAR-2 antibodypolypeptides substantially free of contaminating endogenous materials.

Antigen binding proteins may be prepared, and screened for desiredproperties, by any of a number of known techniques. Certain of thetechniques involve isolating a nucleic acid encoding a polypeptide chain(or portion thereof) of an antigen binding protein of interest (e.g., ananti-PAR-2 antibody), and manipulating the nucleic acid throughrecombinant DNA technology. The nucleic acid may be fused to anothernucleic acid of interest, or altered (e.g., by mutagenesis or otherconventional techniques) to add, delete, or substitute one or more aminoacid residues, for example.

In one aspect, the present invention provides antigen-binding fragmentsof an anti-PAR-2 antibody of the invention. Such fragments can consistentirely of antibody-derived sequences or can comprise additionalsequences. Examples of antigen-binding fragments include Fab, F(ab′)2,single chain antibodies, diabodies, triabodies, tetrabodies, and domainantibodies. Other examples are provided in Lunde et al., 2002, Biochem.Soc. Trans. 30:500-06.

Single chain antibodies may be formed by linking heavy and light chainvariable domain (Fv region) fragments via an amino acid bridge (shortpeptide linker), resulting in a single polypeptide chain. Suchsingle-chain Fvs (scFvs) have been prepared by fusing DNA encoding apeptide linker between DNAs encoding the two variable domainpolypeptides (V_(L) and V_(H)). The resulting polypeptides can fold backon themselves to form antigen-binding monomers, or they can formmultimers (e.g., dimers, trimers, or tetramers), depending on the lengthof a flexible linker between the two variable domains (Kortt et al.,1997, Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng. 18:95-108). Bycombining different V_(L) and V_(H)-comprising polypeptides, one canform multimeric scFvs that bind to different epitopes (Kriangkum et al.,2001, Biomol. Eng. 18:31-40). Techniques developed for the production ofsingle chain antibodies include those described in U.S. Pat. No.4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544, de Graaf etal., 2002, Methods Mol Biol. 178:379-87.

Antigen binding proteins (e.g., antibodies, antibody fragments, andantibody derivatives) of the invention can comprise any constant regionknown in the art. The light chain constant region can be, for example, akappa- or lambda-type light chain constant region, e.g., a human kappa-or lambda-type light chain constant region. The heavy chain constantregion can be, for example, an alpha-, delta-, epsilon-, gamma-, ormu-type heavy chain constant regions, e.g., a human alpha-, delta-,epsilon-, gamma-, or mu-type heavy chain constant region. In oneembodiment, the light or heavy chain constant region is a fragment,derivative, variant, or mutein of a naturally occurring constant region.

Techniques are known for deriving an antibody of a different subclass orisotype from an antibody of interest, i.e., subclass switching. Thus,IgG antibodies may be derived from an IgM antibody, for example, andvice versa. Such techniques allow the preparation of new antibodies thatpossess the antigen-binding properties of a given antibody (the parentantibody), but also exhibit biological properties associated with anantibody isotype or subclass different from that of the parent antibody.Recombinant DNA techniques may be employed. Cloned DNA encodingparticular antibody polypeptides may be employed in such procedures,e.g., DNA encoding the constant domain of an antibody of the desiredisotype. See also Lantto et al., 2002, Methods Mol. Biol. 178:303-16.Moreover, if an IgG4 is desired, it may also be desired to introduce apoint mutation (CPSCP→CPPCP) in the hinge region as described in Bloomet al., 1997, Protein Science 6:407, incorporated by reference herein)to alleviate a tendency to form intra-H chain disulfide bonds that canlead to heterogeneity in the IgG4 antibodies.

Moreover, techniques for deriving antigen binding proteins havingdifferent properties (i.e., varying affinities for the antigen to whichthey bind) are also known. One such technique, referred to as chainshuffling, involves displaying immunoglobulin variable domain generepertoires on the surface of filamentous bacteriophage, often referredto as phage display. Chain shuffling has been used to prepare highaffinity antibodies to the hapten 2-phenyloxazol-5-one, as described byMarks et al., 1992, BioTechnology, 10:779.

In particular embodiments, antigen binding proteins of the presentinvention have a binding affinity (K_(a)) for PAR-2 of at least 10⁶. Inother embodiments, the antigen binding proteins exhibit a K_(a) of atleast 10⁷, at least 10⁸, at least 10⁹, or at least 10¹⁰. In anotherembodiment, the antigen binding protein exhibits a K_(a) substantiallythe same as that of an antibody described herein in the Examples.

In another embodiment, the present invention provides an antigen bindingprotein that has a low dissociation rate from PAR-2. In one embodiment,the antigen binding protein has a K_(off) of 1×10⁻⁴ s⁻¹ or lower. Inanother embodiment, the K_(off) is 5×10⁻⁵ s⁻¹ or lower. In anotherembodiment, the K_(off) is substantially the same as an antibodydescribed herein in the Examples. In another embodiment, the antigenbinding protein binds to PAR-2 with substantially the same K_(off) as anantibody described herein in the Examples.

In another aspect, the present invention provides an antigen bindingprotein that binds at or near the protease cleavage site of human PAR-2.Antigen binding proteins that bind to the protease cleavage site can bemade using any technique known in the art. For example, such antigenbinding proteins can be isolated using the full-length PAR-2 polypeptide(e.g., in a membrane-bound preparation), a soluble extracellular domainfragment of PAR-2, or a smaller fragment of the PAR-2 extracellulardomain comprising or consisting of the protease cleavage site (examplesof which are provided herein). Antigen binding proteins so isolated canbe screened to determine their binding specificity using any methodknown in the art (examples of which are provided herein).

In another embodiment, the present invention provides an antigen bindingprotein that competes for binding to PAR-2 with an antibody disclosedherein. Such competitive ability can be determined by methods that arewell-known in the art, for example by competetion in binding to PAR-2/Fcin a Western blot (or another peptide-based assay), or by competition ina Ca2+ flux assay as described herein. In one aspect, an antigen bindingprotein that competes for binding to PAR-2 with an antibody disclosedherein binds the same epitope as the antibody. In another aspect, theantigen binding protein that competes for binding to PAR-2 with anantibody disclosed herein inhibits proteolytic activation of PAR-2.

In another aspect, the present invention provides an antigen bindingprotein that binds to human PAR-2 expressed on the surface of a celland, when so bound, inhibits PAR-2 signaling activity in the cellwithout causing a significant reduction in the amount of PAR-2 on thesurface of the cell. Any method for determining or estimating the amountof PAR-2 on the surface and/or in the interior of the cell can be used.In one embodiment, the present invention provides an antigen bindingprotein that binds to or near the protease cleavage site of a humanPAR-2 expressed on the surface of a cell and, when so bound, inhibitsPAR-2 signaling activity in the cell without significantly increasingthe rate of internalization of the PAR-2 from the surface of the cell.In other embodiments, binding of the antigen binding protein to thePAR-2-expressing cell causes less than about 75%, 50%, 40%, 30%, 20%,15%, 10%, 5%, 1%, or 0.1% of the cell-surface PAR-2 to be internalized.

In another aspect, the present invention provides an antigen bindingprotein having a half-life of at least one day in vitro or in vivo(e.g., when administered to a human subject). In one embodiment, theantigen binding protein has a half-life of at least three days. Inanother embodiment, the antigen binding protein has a half-life of fourdays or longer. In another embodiment, the antigen binding protein has ahalf-life of eight days or longer. In another embodiment, the antigenbinding protein is derivatized or modified such that it has a longerhalf-life as compared to the underivatized or unmodified antigen bindingprotein. In another embodiment, the antigen binding protein contains oneor more point mutations to increase serum half life, such as describedin WO 00/09560, published Feb. 24, 2000, incorporated by reference.

The present invention further provides multi-specific antigen bindingproteins, for example, bispecific antigen binding protein, e.g., antigenbinding protein that bind to two different epitopes of PAR-2, or to anepitope of PAR-2 and an epitope of another molecule, via two differentantigen binding sites or regions. Moreover, bispecific antigen bindingprotein as disclosed herein can comprise a PAR-2 binding site from oneof the herein-described antibodies and a second PAR-2 binding regionfrom another of the herein-described antibodies, including thosedescribed herein by reference to other publications. Alternatively, abispecific antigen binding protein may comprise an antigen binding sitefrom one of the herein described antibodies and a second antigen bindingsite from another PAR-2 antibody that is known in the art, or from anantibody that is prepared by known methods or the methods describedherein.

Numerous methods of preparing bispecific antibodies are known in theart, and discussed in U.S. patent application Ser. No. 09/839,632, filedApr. 20, 2001 (incorporated by reference herein). Such methods includethe use of hybrid-hybridomas as described by Milstein et al., 1983,Nature 305:537, and others (U.S. Pat. No. 4,474,893, U.S. Pat. No.6,106,833), and chemical coupling of antibody fragments (Brennan et al.,1985, Science 229:81; Glennie et al., 1987, J. Immunol. 139:2367; U.S.Pat. No. 6,010,902). Moreover, bispecific antibodies can be produced viarecombinant means, for example by using leucine zipper moieties (i.e.,from the Fos and Jun proteins, which preferentially form heterodimers;Kostelny et al., 1992, J. Immnol 148:1547) or other lock and keyinteractive domain structures as described in U.S. Pat. No. 5,582,996.Additional useful techniques include those described in Kortt et al.,1997, supra; U.S. Pat. No. 5,959,083; and U.S. Pat. No. 5,807,706.

In another aspect, the antigen binding protein of the present inventioncomprises a derivative of an antibody. The derivatized antibody cancomprise any molecule or substance that imparts a desired property tothe antibody, such as increased half-life in a particular use. Thederivatized antibody can comprise, for example, a detectable (orlabeling) moiety (e.g., a radioactive, colorimetric, antigenic orenzymatic molecule, a detectable bead (such as a magnetic orelectrodense (e.g., gold) bead), or a molecule that binds to anothermolecule (e.g., biotin or streptavidin)), a therapeutic or diagnosticmoiety (e.g., a radioactive, cytotoxic, or pharmaceutically activemoiety), or a molecule that increases the suitability of the antibodyfor a particular use (e.g., administration to a subject, such as a humansubject, or other in vivo or in vitro uses). Examples of molecules thatcan be used to derivatize an antibody include albumin (e.g., human serumalbumin) and polyethylene glycol (PEG). Albumin-linked and PEGylatedderivatives of antibodies can be prepared using techniques well known inthe art. In one embodiment, the antibody is conjugated or otherwiselinked to transthyretin (TTR) or a TTR variant. The TTR or TTR variantcan be chemically modified with, for example, a chemical selected fromthe group consisting of dextran, poly(n-vinyl pyurrolidone),polyethylene glycols, propropylene glycol homopolymers, polypropyleneoxide/ethylene oxide co-polymers, polyoxyethylated polyols and polyvinylalcohols. US Pat. App. No. 20030195154.

In another aspect, the present invention provides methods of screeningfor a molecule that binds to PAR-2 using the antigen binding proteins ofthe present invention. Any suitable screening technique can be used. Inone embodiment, a PAR-2 molecule, or a fragment thereof to which anantigen binding protein of the present invention binds, is contactedwith the antigen binding protein of the invention and with anothermolecule, wherein the other molecule binds to PAR-2 if it reduces thebinding of the antigen binding protein to PAR-2. Binding of the antigenbinding protein can be detected using any suitable method, e.g., anELISA. Detection of binding of the antigen binding protein to PAR-2 canbe simplified by detectably labeling the antigen binding protein, asdiscussed above. In another embodiment, the PAR-2-binding molecule isfurther analyzed to determine whether it inhibits PAR-2 activationand/or signaling.

Nucleic Acids

In one aspect, the present invention provides isolated nucleic acidmolecules. The nucleic acids comprise, for example, polynucleotides thatencode all or part of an antigen binding protein, for example, one orboth chains of an antibody of the invention, or a fragment, derivative,mutein, or variant thereof, polynucleotides sufficient for use ashybridization probes, PCR primers or sequencing primers for identifying,analyzing, mutating or amplifying a polynucleotide encoding apolypeptide, anti-sense nucleic acids for inhibiting expression of apolynucleotide, and complementary sequences of the foregoing. Thenucleic acids can be any length. They can be, for example, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350,400, 450, 500, 750, 1,000, 1,500, 3,000, 5,000 or more nucleotides inlength, and/or can comprise one or more additional sequences, forexample, regulatory sequences, and/or be part of a larger nucleic acid,for example, a vector. The nucleic acids can be single-stranded ordouble-stranded and can comprise RNA and/or DNA nucleotides, andartificial variants thereof (e.g., peptide nucleic acids).

Nucleic acids encoding antibody polypeptides (e.g., heavy or lightchain, variable domain only, or full length) may be isolated fromB-cells of mice that have been immunized with PAR-2. The nucleic acidmay be isolated by conventional procedures such as polymerase chainreaction (PCR).

The invention further provides nucleic acids that hybridize to othernucleic acids under particular hybridization conditions. Methods forhybridizing nucleic acids are well-known in the art. See, e.g., CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. As defined herein, a moderately stringent hybridizationcondition uses a prewashing solution containing 5× sodiumchloride/sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0),hybridization buffer of about 50% formamide, 6×SSC, and a hybridizationtemperature of 55° C. (or other similar hybridization solutions, such asone containing about 50% formamide, with a hybridization temperature of42° C.), and washing conditions of 60° C., in 0.5×SSC, 0.1% SDS. Astringent hybridization condition hybridizes in 6×SSC at 45° C.,followed by one or more washes in 0.1×SSC, 0.2% SDS at 68° C.Furthermore, one of skill in the art can manipulate the hybridizationand/or washing conditions to increase or decrease the stringency ofhybridization such that nucleic acids comprising nucleotide sequencesthat are at least 65, 70, 75, 80, 85, 90, 95, 98 or 99% identical toeach other typically remain hybridized to each other. The basicparameters affecting the choice of hybridization conditions and guidancefor devising suitable conditions are set forth by, for example,Sambrook, Fritsch, and Maniatis (1989, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,chapters 9 and 11; and Current Protocols in Molecular Biology, 1995,Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and6.3-6.4), and can be readily determined by those having ordinary skillin the art based on, for example, the length and/or base composition ofthe DNA.

Changes can be introduced by mutation into a nucleic acid, therebyleading to changes in the amino acid sequence of a polypeptide (e.g., anantigen binding protein) that it encodes. Mutations can be introducedusing any technique known in the art. In one embodiment, one or moreparticular amino acid residues are changed using, for example, asite-directed mutagenesis protocol. In another embodiment, one or morerandomly selected residues is changed using, for example, a randommutagenesis protocol. However it is made, a mutant polypeptide can beexpressed and screened for a desired property (e.g., binding to PAR-2 orblocking the proteolytic activation of PAR-2).

Mutations can be introduced into a nucleic acid without significantlyaltering the biological activity of a polypeptide that it encodes. Forexample, one can make nucleotide substitutions leading to amino acidsubstitutions at non-essential amino acid residues. In one embodiment, anucleotide sequence, or a desired fragment, variant, or derivativethereof, is mutated such that it encodes an amino acid sequencecomprising one or more deletions or substitutions of amino acidresidues. In another embodiment, the mutagenesis inserts an amino acidadjacent to one or more amino acid residues. Alternatively, one or moremutations can be introduced into a nucleic acid that selectively changethe biological activity (e.g., binding of PAR-2, inhibiting proteolyticactivation of PAR-2, etc.) of a polypeptide that it encodes. Forexample, the mutation can quantitatively or qualitatively change thebiological activity. Examples of quantitative changes includeincreasing, reducing or eliminating the activity. Examples ofqualitative changes include changing the antigen specificity of anantigen binding protein.

In another aspect, the present invention provides nucleic acid moleculesthat are suitable for use as primers or hybridization probes for thedetection of nucleic acid sequences of the invention. A nucleic acidmolecule of the invention can comprise only a portion of a nucleic acidsequence encoding a full-length polypeptide of the invention, forexample, a fragment that can be used as a probe or primer or a fragmentencoding an active portion (e.g., a PAR-2 binding portion) of apolypeptide of the invention.

Probes based on the sequence of a nucleic acid of the invention can beused to detect the nucleic acid or similar nucleic acids, for example,transcripts encoding a polypeptide of the invention. The probe cancomprise a label group, e.g., a radioisotope, a fluorescent compound, anenzyme, or an enzyme co-factor. Such probes can be used to identify acell that expresses the polypeptide.

In another aspect, the present invention provides vectors comprising anucleic acid encoding a polypeptide of the invention or a portionthereof. Examples of vectors include, but are not limited to, plasmids,viral vectors, non-episomal mammalian vectors and expression vectors,for example, recombinant expression vectors.

The recombinant expression vectors of the invention can comprise anucleic acid of the invention in a form suitable for expression of thenucleic acid in a host cell. The recombinant expression vectors includeone or more regulatory sequences, selected on the basis of the hostcells to be used for expression, which is operably linked to the nucleicacid sequence to be expressed. Regulatory sequences include those thatdirect constitutive expression of a nucleotide sequence in many types ofhost cells (e.g., SV40 early gene enhancer, Rous sarcoma virus promoterand cytomegalovirus promoter), those that direct expression of thenucleotide sequence only in certain host cells (e.g., tissue-specificregulatory sequences, see Voss et al., 1986, Trends Biochem. Sci.11:287, Maniatis et al., 1987, Science 236:1237, incorporated byreference herein in their entireties), and those that direct inducibleexpression of a nucleotide sequence in response to particular treatmentor condition (e.g., the metallothionin promoter in mammalian cells andthe tet-responsive and/or streptomycin responsive promoter in bothprokaryotic and eukaryotic systems (see id.). It will be appreciated bythose skilled in the art that the design of the expression vector candepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. The expression vectorsof the invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein.

In another aspect, the present invention provides host cells into whicha recombinant expression vector of the invention has been introduced. Ahost cell can be any prokaryotic cell (for example, E. coli) oreukaryotic cell (for example, yeast, insect, or mammalian cells (e.g.,CHO cells)). Vector DNA can be introduced into prokaryotic or eukaryoticcells via conventional transformation or transfection techniques. Forstable transfection of mammalian cells, it is known that, depending uponthe expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., for resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those that confer resistance todrugs, such as G418, hygromycin and methotrexate. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die), among other methods.

Indications

In one aspect, the present invention provides methods of treating asubject. The method can, for example, have a generally salubrious effecton the subject, e.g., it can increase the subject's expected longevity.Alternatively, the method can, for example, treat, prevent, cure,relieve, or ameliorate (“treat”) a disease, disorder, condition, orillness (“a condition”). Among the conditions to be treated inaccordance with the present invention are conditions characterized byinappropriate expression or activity of PAR-2. In some such conditions,the expression or activity level is too high, and the treatmentcomprises administering a PAR-2 antagonist as described herein.

Specific medical conditions and diseases that are treatable orpreventable with the antigen binding proteins of this invention includeinflammatory conditions of the gastrointestinal system, includingcoeliac disease, Crohn's disease; ulcerative colitis; idiopathicgastroparesis; pancreatitis, including chronic pancreatitis;inflammatory bowel disease and ulcers, including gastric and duodenalulcers. The antigen binding proteins of this invention are also usefulin treating or ameliorating inflammatory conditions of the airway, suchas asthma, chronic obstructive pulmonary disease, and the like.

Rheumatic disorders that are treatable with the antigen binding proteinsof this invention include adult and juvenile rheumatoid arthritis;scleroderma; systemic lupus erythematosus; gout; osteoarthritis;polymyalgia rheumatica; seronegative spondylarthropathies, includingankylosing spondylitis, and Reiter's disease, psoriatic arthritis andchronic Lyme arthritis. Also treatable or preventable with thesepolypeptides are Still's disease and uveitis associated with rheumatoidarthritis. In addition, the polypeptide therapies of the invention areused in treating disorders resulting in inflammation of the voluntarymuscle and other muscles, including dermatomyositis, inclusion bodymyositis, polymyositis, and lymphangioleimyomatosis.

The methods described herein can be treated with the antigen bindingproteins of this invention in combination with other cytokines, cytokineinhibitors and reagents (also referred to herein as immunomodulators).For example, IL-18 antagonists; including soluble IL-18 receptor,antibodies to IL-18 or the IL-18 receptor, IL-18 binding protein; TNFinhibitors, including ENBREL®; IL-1 inhibitors, including soluble formsof type II IL-1R, type II IL-1R, antibodies to IL-1, antibodies to typeI IL-1R; and or other active agents that are effective in treating thedisclosed medical conditions and diseases. The compositions and/ormethods of the present invention also can be used, for example, incosmetic treatments, in veterinary treatments, to increase longevity, totreat reproductive defects, and to treat a variety of PAR-2 relateddisorders. In addition, in certain such conditions, the expression oractivity level of PAR-2 is too low, and the treatment comprisesadministering a PAR-2 agonist; such treatments are also comprehendedherein.

Therapeutic Methods and Administration of Antigen Binding Proteins

Certain methods provided herein comprise administering a PAR-2 bindingantigen binding protein to a subject, thereby reducing a PAR-2-inducedbiological response that plays a role in a particular condition. Inparticular embodiments, methods of the invention involve contactingendogenous PAR-2 with a PAR-2 binding antigen binding protein, e.g., viaadministration to a subject or in an ex vivo procedure.

The term “treatment” encompasses alleviation or prevention of at leastone symptom or other aspect of a disorder, or reduction of diseaseseverity, and the like. An antigen binding protein need not effect acomplete cure, or eradicate every symptom or manifestation of a disease,to constitute a viable therapeutic agent. As is recognized in thepertinent field, drugs employed as therapeutic agents may reduce theseverity of a given disease state, but need not abolish everymanifestation of the disease to be regarded as useful therapeuticagents. Similarly, a prophylactically administered treatment need not becompletely effective in preventing the onset of a condition in order toconstitute a viable prophylactic agent. Simply reducing the impact of adisease (for example, by reducing the number or severity of itssymptoms, or by increasing the effectiveness of another treatment, or byproducing another beneficial effect), or reducing the likelihood thatthe disease will occur or worsen in a subject, is sufficient. Oneembodiment of the invention is directed to a method comprisingadministering to a patient a PAR-2 antagonist in an amount and for atime sufficient to induce a sustained improvement over baseline of anindicator that reflects the severity of the particular disorder.

As is understood in the pertinent field, pharmaceutical compositionscomprising the molecules of the invention are administered to a subjectin a manner appropriate to the indication. Pharmaceutical compositionsmay be administered by any suitable technique, including but not limitedto parenterally, topically, or by inhalation. If injected, thepharmaceutical composition can be administered, for example, viaintra-articular, intravenous, intramuscular, intralesional,intraperitoneal or subcutaneous routes, by bolus injection, orcontinuous infusion. Localized administration, e.g. at a site of diseaseor injury is contemplated, as are transdermal delivery and sustainedrelease from implants. Delivery by inhalation includes, for example,nasal or oral inhalation, use of a nebulizer, inhalation of theantagonist in aerosol form, and the like. Other alternatives includeeyedrops; oral preparations including pills, syrups, lozenges or chewinggum; and topical preparations such as lotions, gels, sprays, andointments.

Use of antigen binding proteins in ex vivo procedures also iscontemplated. For example, a patient's blood or other bodily fluid maybe contacted with an antigen binding protein that binds PAR-2 ex vivo.The antigen binding protein may be bound to a suitable insoluble matrixor solid support material.

Advantageously, antigen binding proteins are administered in the form ofa composition comprising one or more additional components such as aphysiologically acceptable carrier, excipient or diluent. Optionally,the composition additionally comprises one or more physiologicallyactive agents, for example, a second inflammation- or immune-inhibitingsubstance, an anti-angiogenic substance, an analgesic substance, etc.,non-exclusive examples of which are provided herein. In variousparticular embodiments, the composition comprises one, two, three, four,five, or six physiologically active agents in addition to a PAR-2binding antigen binding protein

In one embodiment, the pharmaceutical composition comprise an antigenbinding protein of the invention together with one or more substancesselected from the group consisting of a buffer, an antioxidant such asascorbic acid, a low molecular weight polypeptide (such as those havingfewer than 10 amino acids), a protein, an amino acid, a carbohydratesuch as glucose, sucrose or dextrins, a chelating agent such as EDTA,glutathione, a stabilizer, and an excipient. Neutral buffered saline orsaline mixed with conspecific serum albumin are examples of appropriatediluents. In accordance with appropriate industry standards,preservatives such as benzyl alcohol may also be added. The compositionmay be formulated as a lyophilizate using appropriate excipientsolutions (e.g., sucrose) as diluents. Suitable components are nontoxicto recipients at the dosages and concentrations employed. Furtherexamples of components that may be employed in pharmaceuticalformulations are presented in Remington's Pharmaceutical Sciences,16^(th) Ed. (1980) and 20^(th) Ed. (2000), Mack Publishing Company,Easton, Pa.

Kits for use by medical practitioners include anPAR-2-inhibitingsubstance of the invention and a label or other instructions for use intreating any of the conditions discussed herein. In one embodiment, thekit includes a sterile preparation of one or more PAR-2 binding antigenbinding proteins, which may be in the form of a composition as disclosedabove, and may be in one or more vials.

Dosages and the frequency of administration may vary according to suchfactors as the route of administration, the particular antigen bindingproteins employed, the nature and severity of the disease to be treated,whether the condition is acute or chronic, and the size and generalcondition of the subject. Appropriate dosages can be determined byprocedures known in the pertinent art, e.g. in clinical trials that mayinvolve dose escalation studies.

A PAR-2 inhibiting substance of the invention may be administered, forexample, once or more than once, e.g., at regular intervals over aperiod of time. In particular embodiments, an antigen binding protein isadministered over a period of at least a month or more, e.g., for one,two, or three months or even indefinitely. For treating chronicconditions, long-term treatment is generally most effective. However,for treating acute conditions, administration for shorter periods, e.g.from one to six weeks, may be sufficient. In general, the antigenbinding protein is administered until the patient manifests a medicallyrelevant degree of improvement over baseline for the chosen indicator orindicators.

Particular embodiments of the present invention involve administering anantigen binding protein at a dosage of from about 1 ng of antigenbinding protein per kg of subject's weight per day (“1 ng/kg/day”) toabout 10 mg/kg/day, more preferably from about 500 ng/kg/day to about 5mg/kg/day, and most preferably from about 5 μg/kg/day to about 2mg/kg/day, to a subject. In additional embodiments, an antigen bindingprotein is administered to adults one time per week, two times per week,or three or more times per week, to treat a PAR-2 mediated disease,condition or disorder, e.g., a medical disorder disclosed herein. Ifinjected, the effective amount of antigen binding protein per adult dosemay range from 1-20 mg/m², and preferably is about 5-12 mg/m².Alternatively, a flat dose may be administered; the amount may rangefrom 5-100 mg/dose. One range for a flat dose is about 20-30 mg perdose. In one embodiment of the invention, a flat dose of 25 mg/dose isrepeatedly administered by injection. If a route of administration otherthan injection is used, the dose is appropriately adjusted in accordancewith standard medical practices. One example of a therapeutic regimeninvolves injecting a dose of about 20-30 mg of antigen binding proteinto one to three times per week over a period of at least three weeks,though treatment for longer periods may be necessary to induce thedesired degree of improvement. For pediatric subjects (age 4-17), oneexemplary suitable regimen involves the subcutaneous injection of 0.4mg/kg, up to a maximum dose of 25 mg of antigen binding proteinadministered two or three times per week.

Particular embodiments of the methods provided herein involvesubcutaneous injection of from 0.5 mg to 10 mg, preferably from 3 to 5mg, of an antigen binding protein, once or twice per week. Anotherembodiment is directed to pulmonary administration (e.g., by nebulizer)of 3 or more mg of antigen binding protein once a week.

Examples of therapeutic regimens provided herein comprise subcutaneousinjection of an antigen binding protein once a week, at a dose of 1.5 to3 mg, to treat a condition in which PAR-2 signaling plays a role.Examples of such conditions are provided herein and include, forexample, rheumatic conditions as previously described, and otherconditions in which excessive inflammation plays a role (describedherein;

for example, inflammatory bowel disease, pancreatitis, etc). Weeklyadministration of antigen binding protein is continued until a desiredresult is achieved, e.g., the subject's symptoms subside. Treatment mayresume as needed, or, alternatively, maintenance doses may beadministered.

Other examples of therapeutic regimens provided herein comprisesubcutaneous or intravenous administration of a dose of 1, 3, 5, 6, 7,8, 9, 10, 11, 12, 15, or 20 milligrams of a PAR-2 inhibitor of thepresent invention per kilogram body mass of the subject (mg/kg). Thedose can be administered once to the subject, or more than once at acertain interval, for example, once a day, three times a week, twice aweek, once a week, three times a month, twice a month, once a month,once every two months, once every three months, once every six months,or once a year. The duration of the treatment, and any changes to thedose and/or frequency of treatment, can be altered or varied during thecourse of treatment in order to meet the particular needs of thesubject.

In another embodiment, an antigen binding protein is administered to thesubject in an amount and for a time sufficient to induce an improvement,preferably a sustained improvement, in at least one indicator thatreflects the severity of the disorder that is being treated. Variousindicators that reflect the extent of the subject's illness, disease orcondition may be assessed for determining whether the amount and time ofthe treatment is sufficient. Such indicators include, for example,clinically recognized indicators of disease severity, symptoms, ormanifestations of the disorder in question. In one embodiment, animprovement is considered to be sustained if the subject exhibits theimprovement on at least two occasions separated by two to four weeks.The degree of improvement generally is determined by a physician, whomay make this determination based on signs, symptoms, biopsies, or othertest results, and who may also employ questionnaires that areadministered to the subject, such as quality-of-life questionnairesdeveloped for a given disease.

Elevated levels of PAR-2 and/or activation of PAR-2 are associated witha number of disorders, including, for example, inflammatory conditionsof the skin, joints, gastrointestinal system and/or airway. Subjectswith a given disorder may be screened, to identify those individuals whohave elevated PAR-2 activation, thereby identifying the subjects who maybenefit most from treatment with a PAR-2 binding antigen bindingprotein. Thus, treatment methods provided herein optionally comprise afirst step of measuring a subject's PAR-2 activation levels. An antigenbinding protein may be administered to a subject in whom PAR-2activation is elevated above normal.

A subject's levels of PAR-2 activity may be monitored before, duringand/or after treatment with an antigen binding protein, to detectchanges, if any, in PAR-2 activity. For some disorders, the incidence ofelevated PAR-2 activity may vary according to such factors as the stageof the disease or the particular form of the disease. Known techniquesmay be employed for measuring PAR-2 activity, e.g., in a subject'sserum, blood or tissue samples. PAR-2 activity may be measured using anysuitable technique.

Particular embodiments of methods and compositions of the inventioninvolve the use of an antigen binding protein and one or more additionalPAR-2 antagonists, for example, two or more antigen binding proteins ofthe invention, or an antigen binding protein of the invention and one ormore other PAR-2 antagonists. In further embodiments, antigen bindingprotein are administered alone or in combination with other agentsuseful for treating the condition with which the patient is afflicted.

Examples of such agents include both proteinaceous and non-proteinaceousdrugs. When multiple therapeutics are co-administered, dosages may beadjusted accordingly, as is recognized in the pertinent art.“Co-administration” and combination therapy are not limited tosimultaneous administration, but also include treatment regimens inwhich an antigen binding protein is administered at least once during acourse of treatment that involves administering at least one othertherapeutic agent to the patient.

Examples of other agents that may be co-administered with an antigenbinding protein are other antigen binding proteins or therapeuticpolypeptides that are chosen according to the particular condition to betreated. Alternatively, non-proteinaceous drugs that are useful intreating one of the particular conditions discussed above may beco-administered with a PAR-2 antagonist.

Combination Therapy

In another aspect, the present invention provides a method of treating asubject with a PAR-2 inhibiting antigen binding protein and one or moreother treatments. In one embodiment, such a combination therapy achievessynergy or an additive effect by, for example, attacking multiple sitesor molecular targets in a tumor. Types of combination therapies that canbe used in connection with the present invention include inhibiting oractivating (as appropriate) multiple nodes in a single disease-relatedpathway, multiple pathways in a target cell, and multiple cell typeswithin a target tissue.

In another embodiment, a combination therapy method comprisesadministering to the subject two, three, four, five, six, or more of thePAR-2 agonists or antagonists described herein. In another embodiment,the method comprises administering to the subject two or more treatmentsthat together inhibit or activate (directly or indirectly)PAR-2-mediated signal transduction. Examples of such methods includeusing combinations of two or more PAR-2 inhibiting antigen bindingproteins, of a PAR-2 inhibiting antigen binding protein and one or moreother therapeutic moiety having anti-inflammatory properties (forexample, non-steroidal anti-inflammatory agents, steroids, and/orimmunomodulators), or of a PAR-2 inhibiting antigen binding protein andone or more other treatments (e.g., surgery, ultrasound, or treatmenteffective to reduce inflammation). Furthermore, one or more anti-PAR-2antibodies or antibody derivatives can be used in combination with oneor more molecules or other treatments, wherein the other molecule(s)and/or treatment(s) do not directly bind to or affect PAR-2, but whichcombination is effective for treating or preventing the condition beingtreated. In one embodiment, one or more of the molecule(s) and/ortreatment(s) treats or prevents a condition that is caused by one ormore of the other molecule(s) or treatment(s) in the course of therapy,e.g., nausea, fatigue, alopecia, cachexia, insomnia, etc. In every casewhere a combination of molecules and/or other treatments is used, theindividual molecule(s) and/or treatment(s) can be administered in anyorder, over any length of time, which is effective, e.g.,simultaneously, consecutively, or alternately. In one embodiment, themethod of treatment comprises completing a first course of treatmentwith one molecule or other treatment before beginning a second course oftreatment. The length of time between the end of the first course oftreatment and beginning of the second course of treatment can be anylength of time that allows the total course of therapy to be effective,e.g., seconds, minutes, hours, days, weeks, months, or even years.

In another embodiment, the method comprises administering one or more ofthe PAR-2 antagonists described herein and one or more other treatments(e.g., a therapeutic or palliative treatment). Where a method comprisesadministering more than one treatment to a subject, it is to beunderstood that the order, timing, number, concentration, and volume ofthe administrations is limited only by the medical requirements andlimitations of the treatment, i.e., two treatments can be administeredto the subject, e.g., simultaneously, consecutively, alternately, oraccording to any other regimen.

The following examples, both actual and prophetic, are provided for thepurpose of illustrating specific embodiments or features of the instantinvention and do not limit its scope.

EXAMPLE 1 Preparation of Monoclonal Antibodies

PAR-2 polypeptides may be employed as immunogens in generatingmonoclonal antibodies by conventional techniques, e.g., techniquesdescribed in U.S. Pat. No. 5,599,905, hereby incorporated by reference.It is recognized that polypeptides in various forms may be employed asimmunogens, e.g., full length proteins, fragments thereof, fusionproteins thereof such as Fc fusions, cells expressing the recombinantprotein on the cell surface, etc.

To summarize an example of such a procedure, a loop 1 peptide of PAR-2(TNRSSKGRSLIGKVDGTS; amino acids 29 through 46 of SEQ ID NO:2), havingan additional C-terminal cysteine residue to facilitate conjugation, isconjugated to maleiimide-activated keyhole limpet hemocyanin (KLH;obtainable for example from Pierce Biotechnology Inc., Rockford, Ill.)to yield a PAR-2 immunogen. For a first immunization, 100 micrograms ofimmunogen (containing 50 micrograms of peptide) is emulsified incomplete Freund's adjuvant (CFA) at 1:1 ratio by volume and injectedsubcutaneously in a final volume of 200 microliters for each mouse.

Immunized animals are boosted three to four more times with additionalimmunogen to increase the antigen-specific response, at intervals of twoto four weeks (although longer intervals may be employed. For example, asecond injection of 50 micrograms of immunogen (containing 25 microgramsof peptide) mixed with incomplete Freund's adjuvant in a final volume of200 ul is injected subcutaneously into each mouse about four weeks daysafter the primary immunization. A third injection (20 micrograms ofimmunogen containing 10 micrograms of peptide mixed with an adjuvantsuch as Ribi adjuvant) may be given by subcutaneous and/orintraperitoneal route from about 1 days after the second injection. Ifdesired, a fourth injection (20 micrograms of immunogen containing 10micrograms of peptide mixed with incomplete Freund's adjuvant) may begiven by subcutaneous and/or intraperitoneal route from about 14 toabout 28 after the third injection. A final injection is given, usuallyabout five days prior to fusion, utilizing 50 micrograms of immunogencontaining 25 micrograms of peptide in PBS, by intraperitonealinjection.

Serum samples may be periodically taken by retro-orbital bleeding ortail-tip excision for testing by peptide ELISA (enzyme-linkedimmunosorbent assay), or another suitable assay, to evaluate antibodytiter. At the time of fusion, the animals are sacrificed, splenocytesharvested, and fused to the murine myeloma cell line SP2/O (ATCC CRL1581). The resulting hybridoma cell lines are plated in multiplemicrotiter plates in a HAT selective medium (hypoxanthine, aminopterin,and thymidine) to facilitate proliferation of spleen cell-myeloma hybridcells.

Hybridoma clones thus generated are screened for reactivity with PAR-2.Initial screening of hybridoma supernatants may utilize a peptide ELISA,a whole cell ELISA and/or a cell-based assay suitable forhigh-throughput screening (fluorometric microvolume assay technology orFMAT, substantially as described by Fiscella, et al., NatureBiotechnology 21:302-307 (2003). Hybridomas that are positive in thisscreening method may be further cultured to provide larger amounts ofantibody, which can then be purified as described below and screened byadditional cell-based assay(s) (for example, a flash plate assay usingcalls cells co-expressing apoaequorin, a Ca2+-sensitive photoprotein,substantially as described by Le Poul et al., J. Biomol. Screen.7(1):57-65; 2002) and PAR-2, or a fluorometric imaging plate reader(FLIPR) assay, which is used to determine changes in intracellular Ca2+levels, substantially as described in S. Pitchford, Genetic EngineeringNews vol. 18, Number 15 (1998) and/or Sullivan et al., Methods inMolecular Biology vol. 114, pp 125-133 (1999), which are incorporated byreference herein.

Selected hybridomas can be further cloned and tested to ensure stableproduction monoclonal antibody. Hybridomas can be cultured in vitro, orpass aged as ascites fluid in suitable host mammals. The resultingmonoclonal antibodies may be purified by ammonium sulfate precipitationfollowed by gel exclusion chromatography, and/or affinity chromatographybased on binding of antibody to Protein G, for example. Severalhybridomas were generated and tested for binding in a whole-cell ELISAusing PAR-2 expressing cells, and in a flash plate assay; results areshown in Table 1 below.

TABLE 1 Blocking in Clone ID Binding FLASH #47 + + #21 + N.A. #48 + +#6  + + #15 + N.A. #13 + +/− #49 + + #10 + − #46 + +/−

EXAMPLE 2 Purification of Anti-PAR2 Hybridoma Antibodies for Screening

Hybridoma cells are cultured for a time and under conditions to yield asample of about 35 ml of hybridoma supernatant fluid. To each sample isadded 12 ml of 4×-Protein A Binding Buffer (1.6 M citric acid, 100 mMtris, pH 9.15) and about 300 μl of a 67% slurry of MabSelect™ Media (GEHealthcare, Piscataway, N.J.). The resulting slurry is rotated gentlyover night at 4° C.

After overnight incubation, the samples are centrifuged to sediment theresin and the monoclonal antibodies bound thereto, for example at 2,000RPM in a G3.8 centrifuge rotor (Beckman Coulter, Fullerton, Calif.) for5 minutes at 4° C. with no brake. All but about 300 μl of thesupernatant fluid is removed and the resin is resuspended to form aconcentrated slurry.

The concentrated slurry is transferred to a microcentrifuge tube andsufficient 1×-Protein A Binding Buffer (400 mM citric acid, 25 mM tris,pH 8.9) is added to bring the total volume up to about 1 ml. The slurryis resuspended, then centrifuged at about 14,000 g for 5 seconds. Thesupernatant fluid is removed from the resulting pellet, which is washeda total of three times in a similar manner (i.e. by resuspending inabout 1 ml of 1×-Protein A Binding Buffer, centrifuging, removingsupernatant and resuspending in fresh buffer).

After three washes, the pellet is resuspended in 400 μl Elution Buffer(200 mM formic acid) and agitated for 10 min at room temperature, thencentrifuged at 14,000 g for 5 seconds. The supernatant is carefullyremoved as eluate, and the pellet is eluted again in a manner similar tothat described above for a total of three elution cycles. The eluatesfrom the three elution cycles are combined, centrifuge at 14,000 g for 5min room temperature and transferred to a fresh tube. The pH is adjustedto 7.8-8.2 by adding 2 M tris base (235 mM_(f)) and mixing quickly. Thesamples are again centrifuged at 14,000 g for 5 min at room temperature,and designated as pH Shift Soluble. A spectral scan of each sample(diluted by adding 20 μl of the sample to 700 μl water) is run from 250to 350 nm, and protein concentration is verified by loading 0.5 μg eachantibody-containing sample on a reducing 4-20% SDS-PAGE gel with anappropriate antibody standard.

EXAMPLE 3 Purification and Western Blot of PAR-2/Fc polypeptide

Full length N-terminal PAR-2/Fc polypeptide (SEQ ID NO:5) is expressedin CHO cells. Expression supernatant from CHO expression cells culturedin serum-free media contain a CHO cell trypsin-like serine protease thatcleaves PAR-2/Fc at the activation Arg-Ser bond, generating the“clipped” version of the PAR-2/Fc polypeptide. CHO expression cellscultured in 10% fetal calf serum (which contains normal levels of plasmaproteinase inhibitors at concentrations far in excess of theconcentration of the CHO cell trypsin-like serine protease) express fulllength N-terminal PAR-2/Fc in culture supernatants. Both clipped andfull length proteins are purified using MabSelect™ resin substantiallyas previously described (see Example 2). The resultant purifiedFc-constructs are analyzed by amino terminal sequence analysis (Edmandegradation), size exclusion chromatrography, absorbance spectral scan,and mass spectroscopy.

Various amounts of purified full length and clipped N-terminal PAR2-Fcare subjected to SDS-PAGE using 8-16% polyacrylamine gradient gels(Novex gels, Invitrogen Life Technologies) in a Tris-Glycine buffersystem. Gel lanes containing See Blue standards (Novex, Invitrogen LifeTechnologies) for molecular weight identification are also included.Following electrophoresis, proteins are transferred from gels ontonitrocellulose membranes using a Novex XCell II Blot Module (InvitrogenLife Technologies). Membranes are blocked with 1:1, Odyssey blockingbuffer, OBB, (LI-COR Biosciences):TBS (Tris Buffer Saline) overnight at4 C with shaking. Antibodies to be analyzed are diluted in 1:1 OBB:TBSat a desired final concentration for 1 hr at room temperature. Membranesblots are washed extensively with 0.1% Tween 20 in TBS (3-4 changes of100 ml over ˜1 hr). Membranes are then exposed to the appropriatesecondary antibody-Alexa680 (Molecular Probes, Invitrogen LifeTechnologies) conjugate (goat anti-rabbit IgG, or goat anti-mouse IgG)diluted 1:5000 in 1:1 (OBB:TBS) for 1 hr at room temperature. Membranesare washed as described above, and if desired, analyzed using a LI-COROdyssey Infrared Imaging System (LI-COR Biosciences).

EXAMPLE 4 Comparison of PAR-2 Antibodies

Several PAR-2 antibodies were tested in different assay formats; Table 2summarizes the results.

TABLE 2 IC50 Binding to Cleavage site Clone ID # (pre-cloning) Up streamDown stream 47 8 nM xxx — 49 35 nM xx — 6 40 nM xx — 13 50 nM xx — 15 50nM xx — 48 60 nM xx — 10 >1 mM xx xxx 46 >0.5 mM xxx xx

IC50 values were determined in a FLIPR assay for Ca2+ mobilizationdescribed previously; binding to the upstream region of the cleavagesite versus the downstream region of the cleavage site was determinedusing the Western blot assay described previously. More detailed resultsfor a selected antibody are shown in FIG. 1. FIG. 2 presents Westernblot results in which various antibodies were analyzed by Western blotagainst the cleaved (denoted ‘clip’) PAR-2/Fc and the full-length(denoted ‘FL’) PAR-2/Fc. FIG. 3 presents additional Western blotresults, and also compares the characteristics of two monoclonalantibodies that present a contrast in recognition of full-length versusclipped PAR-2/Fc, which as detailed on the slide corresponds withability to antagonize PAR-2. Thus, Clone 10 was weakly antagonistic in aflash plate assay, bound to cells expressing human PAR-2 (HCT116 cells),and to PAR-2-transfected CHO cells via FACS, but not to CHO parentalcells, and bound to both clipped and full-length versions of PAR-2/Fc byWestern blot. In contrast, Clone 33 was strongly antagonistic in a flashplate assay, bound to cells expressing human PAR-2 (HCT116 cells), andto PAR-2-transfected CHO cells via FACS, but not to CHO parental cells,and bound to the full-length version of PAR-2/Fc only by Western blot.

Each reference cited herein is incorporated by reference in its entiretyfor all that it teaches and for all purposes.

What is claimed is:
 1. An isolated antigen binding protein, that bindsto proteinase activated receptor-2 (PAR-2) and antagonizes theproteolytic activation thereof.
 2. The isolated antigen binding proteinof claim 1, wherein the antigen binding protein antagonizes theproteolytic activation of PAR-2 with an IC50 of 60 nM or less in afluorometric imaging plate reader (FLIPR) assay using HCT-116 cells. 3.The isolated antigen binding protein of claim 1 or claim 2, thatspecifically binds to the PAR-2 of a non-human primate, a cynomologousmonkey, a chimpanzee, a non-primate mammal, a rodent, a mouse, a rat, ahamster, a guinea pig, a cat, or a dog.
 4. The isolated antigen bindingprotein of claim 1 or claim 2 wherein said antigen binding proteincomprises: a. a human antibody; b. a humanized antibody; c. a chimericantibody; d. a monoclonal antibody; e. a polyclonal antibody; f. arecombinant antibody; g. an antigen-binding antibody fragment; h. asingle chain antibody; i. a diabody; j. a triabody; k. a tetrabody; l. aFab fragment; m. a F(ab′)₂ fragment; n. a domain antibody; o. an IgDantibody; p. an IgE antibody; q. an IgM antibody; r. an IgG1 antibody;s. an IgG2 antibody; t. an IgG3 antibody; u. an IgG4 antibody; or v. anIgG4 antibody having at least one mutation in a hinge region thatalleviates a tendency to form intra-H chain disulfide bond.
 5. Anisolated cell that secretes an antigen binding protein of claim 1,wherein the cell is a hybridoma.
 6. A method of making an antigenbinding protein that binds PAR-2, comprising incubating the isolatedcell of claim 5 under conditions that allow it to express the antigenbinding protein.
 7. A pharmaceutical composition comprising the antigenbinding protein of claim
 1. 8. A method of treating a condition in asubject comprising administering to the subject the pharmaceuticalcomposition of claim 7, wherein the condition is treatable by reducingthe activity of PAR-2 in the subject.
 9. The method of claim 8 whereinthe subject is a human being.
 10. The method of claim 9 wherein saidcondition is selected from the group consisting of inflammatoryconditions of the skin, joints, gastrointestinal system and/or airway.11. A method of decreasing PAR-2 activity in a subject in need thereofcomprising administering to the subject said pharmaceutical compositionof claim
 7. 12. The method of claim 11 wherein the PAR-2 activity thatis decreased is PAR-2 signalling.
 13. The method of claim 12, whereinthe PAR-2 signalling decrease results from inhibition of proteolyticactivation of PAR-2.